It is well established that the transformation of normal tissue cells to tumor cells is associated with a change of carbohydrate structures (glycoconjugates) on the cell surface. These new or modified glycoconjugates may serve as antigens and may then represent a type of so-called tumor-associated antigens. The cell surface carbohydrates are linked either to a lipid moiety, in which case they are called glycolipids, or to peptides (proteins), in which case they are called glycopeptides (glycoproteins).
Tumor-associated glycoconjugate antigens are previously known in relation to human cancer disease. In melanomas the carbohydrate structures disialosylgangliotetraose and disialosyllactose have been identified linked to a lipid portion. Two carcinoma associated antigens CEA (carcinoembryonal antigen) and GICA (gastro-intestinal cancer antigen) have been demonstrated particularly in gastrointestinal carcinomas while a third antigen, CA-50, seems to have a more general distribution. All the antigens are shedded from the cell surface or secreted from the tumor cell and can be demonstrated in blood serums by means of immunological methods.
The present invention is based on the discovery that patients with certain forms of lung cancer (small cell carcinomas or oat-cell carcinomas) express in the primary lung tumor and its metastases an acidic glycolipid, which is not expressed in normal lung tissue. This glycolipid antigen is a ganglioside with the complete chemical structure Fuc.alpha.1-2Gal.beta.1-3GalNAc.beta.1-4(NeuAc.alpha.2-3)Gal.beta.1-4Glc or IV.sup.2 Fuc.alpha.-,II.sup.3 NeuAc.alpha.-GgOse.sub.4 with the short designation Fuc-GM1 (IUPAC-IUB Lipid Document, 1977). This ganglioside was first isolated from bovine brain (Ghidoni et al., J. Neurochem. 27, 511-515, 1976) but indications for its occurrence in other mammalian organs as adipose tissue, spleen and testis have also been presented. Hakomori and co-workers (Baumann et al, Cancer Res. 39, 2637-2643), 1979) isolated from rat hepatomas a ganglioside, which was tentatively identified as Fuc-GM1. A definitive establishment of the chemical structure of this ganglioside was performed by Svennerholm and collaborators (Fredman et al., Eur. J. Biochem. 116, 553-564) on gangliosides isolated from porcine nervous tissue. They also showed that chronic chloroquine treatment of the pig caused an increased lysosomal storage of Fuc-GM1 in nervous tissue (Klinghardt et al., J. Neurochem. 37, 897-908, 1981) but also in several other organs (Fredman et al., Biochem. J. 201, 581-588, 1982). Although this ganglioside has a wide distribution in pig, we have not been able to show its occurrence in normal human lung tissue or in carcinomas from a large number of human organs. In patients, died in lung small cell carcinoma, all lung tumors contained Fuc-GM1 ganglioside, and in other patients Fuc-GM1 was shed to blood serum. With the assay method for Fuc-GM1 developed by us the tumor antigen was detected on the blood serum in patients with lung small cell carcinoma but not in the blood serum of a control population or of patients with other forms of cancer.
Within the scope of the present invention procedures have been elaborated for the isolation of the tumor associated glycolipid, fucosylsialosylgangliotetraosylceramide with the short designation, ganglioside Fuc-GM1, in pure form from human cancer tissue and from animal organ. The purified ganglioside has by a special device obtained an increased immunogenic activity and then been used for the production of monoclonal antibodies. Procedures have, within the scope of the invention, been developed for the identification and quantitative determination in tissues, tissue sections, cytological preparations and various body fluids. All of these methods for diagnostic purposes are based on the fact that the antigen can be detected and quantitatively determined by the reaction with specific antibodies produced against the pure ganglioside antigen, Fuc-GM1.
The following non-limiting examples illustrate the various sources where the ganglioside antigen can be measured: surgical or autopsy material of lung, liver, brain or similar organs, cytological specimens obtained by biopsy of lung or other organs, cytological specimens obtained by biopsy of lung or other organs, where signs of cancer metastases can be suspected, cells, obtained by aspirations of the respiratory tract, expectorations, or by puncture of pleural or abdominal cavity, biological fluids like whole blood, blood plasma or serum, lymph, cerebrospinal fluid, urine, saliva or similar.
The antigen which is used in the present invention is termed, fucosylsialosylgangliotetraose and has the following chemical structure: Fuc.alpha.1-2Gal.beta.1-3GalNAc.beta.1-4(NeuAc.alpha.2-3)Gal.beta.1-4Glc or IV.sup.2 .alpha.Fucll.sup.3 .alpha.NeuAcGgOse.sub.4 (IUPAC-IUB Lipid Document, 1977). Within the scope of the present invention all antigens, which have been modified by the removal of the terminal glucose moiety or by the substitution of the sialic acid (NeuAc) moiety to the N-acetylgalactosamine (GalNAc) moiety instead of to the galactose (Gal) moiety are covered by this patent application. The monoclonal antibodies referred to in the present invention have all been produced from antigens bound to a lipid group (acylsphingosine) but this claim will also include the binding of the antigenic group to every other compound.
The antigen which has been used in the present invention can be isolated from tumor tissue, obtained by surgery or autopsy of patients with lung small cell carcinoma. The concentration of the antigen in the tumor tissue is in general relatively low, particularly when the patient has been treated with cytostatic drugs, and the isolation of the antigen will then be laborious. We have also found another biological material, which can be used for the isolation of the cancer antigen. When a certain strain of miniature pig is foddered with chloroquine in the diet during 3-6 months, high concentrations of the ganglioside antigen is formed in the pig's central nervous system, particularly in the dorsal root ganglia. The cancer associated ganglioside antigen can then be relatively simply isolated from brain and dorsal root ganglia. By this device one is not limited for the isolation of the antigen to surgery or autopsy material and a large number of practical and ethical difficulties are avoided.
It has previously been found that gangliosides--it means glycolipids containing sialic acid--are poor immunogens and it has been difficult to produce antibodies with high affinity and specificity against them. This difficulty has been circumvented in the present invention by the adsorption of the ganglioside antigen to a bacterial membrane with known high immunogenic activity. By this device a large number of monoclonal antibodies with high specificity and affinity against the cancer antigen have been produced.
The present invention concerns in accordance herewith a procedure for the induction of the cancer associated antigen fucosylsialosylgangliotetraose in the nervous system of a particular type of miniature pig, isolation of the antigen with chromatographical methods and production of monoclonal antibodies against the purified antigen adsorbed to a certain bacterial membrane. The invention includes also the use of the compound fucosylsialosylgangliotetraose and all the derivates of this substance and all types of antibodies directed against them for the diagnosis of different cancer forms, specially certain forms of lung cancer.
It has previously been reported that monoclonal antibodies against certain tumor-associated antigens can suppress tumor development of human tumors implanted in nude mice and clinical studies in patients have also suggested the possibility to suppress or slow down tumor growth in humans (e.g. Herlin, D and Koprowski, H, PNAS (USA), 79:4761-4765, 1982; Adams, D O et al, PNAS (USA) 81:3506-3510, 1984; Sears, H F et al, Lancet I:762-765, 1982). In some of these investigations as well as in other studies, monoclonal antibodies and polyclonal antisers against tumor-associated antigens, in conjunction with macrophages and/or the complement system, have also been found to kill tumor cells in vitro. In no instance, however, has the use of antibodies directed against fucosylsialosylgangliotetraose antigen, which is the subject matter of this invention, been described for any such purposes. Likewise, neither has the fucosylsialosylgangliotetraose antigen been used to induce protective anti-tumor immunity, or antibodies against this antigen been described for detection and localization of tumors using "imaging" techniques which are otherwise obvious possible applications for tumor-associated antigens and tumor-specific antibodies, respectively (e.g. Holmgren J, ed., Tumor marker antigens, Studentlitteratur/Chartwell-Bratt Ltd, Lund, Sweden/Bromley, England, 1985). The present invention also specifically concerns the use of the compound fucosylsialosylgangliotetraose and all the derivatives of this substance and all types of antibodies directed against them for the treatment of small cell lung cancer and other human cancer disease in patients. Such use of the antibodies and antigens in question also extends to the in vivo search for and localization of Fuc-GM1 containing cancers in patients using "imaging" techniques, and the prevention of Fuc-GM1 containing human cancers by stimulating the formation of protective anti-tumor immunity in an individual.